The present invention relates to a composition for topical use containing a ligand for peripheral benzodiazepine receptors.
The invention relates to the use of a substance which binds specifically to the peripheral benzodiazepine receptor (PBR) for the manufacture of a composition for the prophylaxis or treatment of cutaneous stress.
The invention also relates to compositions containing these substances. These compositions may be cosmetic or pharmaceutical, and in particular topical dermatological compositions.
The term xe2x80x9ccutaneous stressxe2x80x9d means the various situations which may cause damage in particular to the epidermis, irrespective of the agent causing this damage. This agent may be inside and/or outside the body, for instance a chemical or free-radical agent or alternatively an external agent such as ultraviolet radiation.
The composition according to the invention is thus intended to prevent and combat skin irritation, dry patches, erythema, dysesthetic sensations, sensations of heating, pruritus of the skin and/or mucous membranes, and ageing, and may also be used in skin disorders such as, for example, psoriasis, pruriginous diseases, herpes, photodermatitis, atopic dermatitis, contact dermatitis, lichens, prurigo, pruritus, insect bites, in fibrosis and other disorders of collagen maturation, in immunological disorders or in dermatological conditions such as eczema.
The PBR ligand, also referred to as xe2x80x9csubstancexe2x80x9d, contained in the composition may be a non-peptide compound, a peptide, a cell extract or tissue extract of animal or plant origin or a product obtained by fermenting a microorganism, for example fermenting a bacterium or fungus.
Many PBR ligands are disclosed in the literature (FIG. 7). Examples which may be mentioned include Ro 5-4864 or chlorodiazepam, Ro 5-2807 or diazepam and PK 11195, or reference may be made to the article Peripheral Benzodiazepine Receptors, Ch. III, J. J. Bourguignon, Ed. E. Giesenxe2x80x94Crouse, Academic Press.
PBR is an 18-kd protein located on the outer membrane of the mitochondria of peripheral tissues. It consists of five transmembrane domains and of a carboxy-terminal portion directed towards the cytosol. Several functions are attributed to PBR depending on the nature of the tissue under consideration: regulation of steroidogenesis, biosynthesis of heme, cell differentiation and growth, control of mitochondrial respiration (Krueger K E, Biochimica and Biophysica Acta 1995, 1241, 453-470). Although its precise function has not yet been fully elucidated, several recent experimental data suggest that PBR might play a fundamental role in regulating the processes of programmed cell death and in protection against free radicals.
It has been shown that PBR is in fact closely associated at the mitochondrial level with apoptosis regulatory proteins such as Bcl2 which prevents rupture of the mitochondrial membrane potential, thus preventing the apoptosis induced in particular by the production of reactive oxygenated radicals (Marchetti P. et al., J. Exp. Med. 1996, 184, 1155-1160); (Marchetti P. et al., J. Immunol. 1996, 157, 4830-4836).
In the context of the present invention, the protective role of PBR against free radicals was directly observed on cells of hematopoietic origin for which a close correlation between the PBR density and the protection against free radicals was demonstrated. Furthermore, in this same study, it was demonstrated that the transfection of PBR into cells lacking this receptor gives protection against the damage caused by oxygenated species (Carayon P. et al., Blood 1996, 87, 3170-3178).
Several literature data suggest that PBR might play an important role in regulating apoptosis processes and in protecting cells against damage caused by free radicals.
Recent phylogenic studies reinforce this novel notion that PBR acts as an apoptosis modulator involved in antioxidant functions. The reason for this is that significant similarities exist between PBR and the protein CrtK of Rhodobacter sphaeroides, a photo-synthetic bacterium. This bacterial protein which functions as a photosensitive oxygen detector, regulates the expression of the genes involved in photosynthesis in response to environmental changes in oxygen tension and in light intensity. The comparison between PBR and CrtK reveals 35% identity and a conservation of sequence between these two proteins which diverged in the phylogeny two billions years ago. This homology suggests a highly specialized and conserved function of PBR which appears to be similar to that of CrtK in the bacterium. Specifically, it has recently been demonstrated that mammalian PBR transfected into Rhodobacter CrtK mutants complements the oxygen-detecting function of CrTK. Thus, this study suggests a key role of PBR in the transduction of oxygen-dependant signals (Yeliseev A A., et al., Proc. Natl. Acad. Sci. 1997, 94, 5101-5106).
However, to date, no substance has ever been precisely indicated as a specific ligand for cutaneous PBR receptors, which is all the more reason why no topically active substance which binds specifically to the PBR receptors has ever been disclosed in the literature.
It has now been shown, in the context of the present invention, that PBR is abundantly expressed in the skin within the various cell compartments of which it is composed: keratinocytes, Langerhans cells, hair follicles and endothelial cells of the dermal vascular system. In the skin, the expression of PBR follows an increasing gradient from the basal layer to the horny layer. This noteworthy organization which favors the differentiated cells of the epidermis that are the most exposed to external stresses is undoubtedly of a primordial physiological importance for protecting the most vulnerable areas of the epidermis. Subcellular studies performed by confocal microscopy indicate, as expected, a colocalization of PBR with Bcl2 in the mitochondrian. Histological studies on skin sections have revealed a surprising distribution of PBR (FIGS. 1 and 2).
Specifically, the expression of this receptor in the epidermis follows a gradient of increasing density from the basal layers to the most differentiated layers of keratinocytes. This highly organized spatial distribution which favors, in terms of density, the outermost and thus the most exposed cells of the epidermis, leads to the assumption that PBR in the skin might represent a natural protection system against free radicals generated by exposure to ultraviolet radiation. The concomitant observation that the distribution of the anti-apoptotic protein Bcl2 obeys a strictly inverse gradient suggests a compensatory role of PBR in preserving the cells that are most differentiated.
This set of data which suggest a protective function of PBR, more particularly in the epidermis, has led to the discovery of natural or synthetic ligands, showing that their interaction with PBR could be beneficial in various situations of cutaneous stress induced by chemical or free-radical agents or alternatively following an exposure to UV.
Thus, according to one of its aspects, the present invention relates to the use of a ligand which is specific for PBR, Ro 5-4864, in cutaneous stress. This ligand is a PBR agonist.
According to another aspect of the invention and on the basis of these observations, a screening directed toward finding natural PBR ligands was undertaken and made it possible to isolate several fractions capable of interacting with this receptor. The potentially protective effect of these natural ligands was then evaluated in various tests inducing a cutaneous stress and in particular in tests of cutaneous erythema induced by UV irradiation. Radical-scavenging properties and skin repair capacities were also investigated.
Biochemical and pharmacological tests were used to demonstrate the activity and advantage of the substances in various situations of cutaneous stress.
The tests performed with PBR were aimed at showing its potential involvement in regulating apoptotic processes and in preserving skin cells against various deleterious stress situations.